脊髓背角TRPM3通过调控NR2B和PSD-95相互作用介导病理性疼痛的机制研究

The spinal dorsal horn is the primary transfer station for accepting and modulating pain signal, and its central sensitization and hyper-excitability play a crucial role in neuropathic pain. Due to the lacking of the understanding of the mechanism of such sensitization, the treatment of neuropathic pain is still a clinical dilemma. Transient receptor potential melastatin 3 (TRPM3) is a new type of noceptive receptor related to heat and we recently discovered that TRPMs in the spinal dorsal horn might play critical role in neuropathic pain. TRPM3 is constitutively expressed in spinal neurons, and gene silencing of spinal TRPM3 not only attenuates behavioral hypersensitivity, but also inhibits interactions between NMDA receptor 2B subunit (NR2B) and post-synaptic density-95 (PSD-95), suggesting spinal TRPM3 may be involved in the development of neuropathic pain via modulating NR2B/PSD-95 interaction. The present proposal hypothesizes that spinal TRPM3 mediates the formation and transmission of nociceptive signal via modulating NR2B/PSD-95 interaction, activating ERK/CREB signaling pathway, affecting neuronal activity, differentiation, and synaptic remolding. We intend to characterize the expression profile in the spinal dorsal horn. Secondly, the role of spinal TRPM3 will be determined with gene silencing and pharmacological techniques. Thirdly, the possible mechanism(s) of spinal TRPM3 modulating persist pain will be explored by various methods, such as disrupting the interaction of spinal NR2B with PSD-95, and CREB antisense technique. The present proposal reveals scientific understanding of the role of TRPM3 in persist pain signal transduction using molecular techniques (RNAi and up-regulating) to explore NR2B/PSD-95 interaction both in vivo and in situ and correlate with behavioral changes in rat neuropathic pain models. The proposal is of great significance in revealing the spinal mechanisms underlying neuropathic pain to guide searching novel therapeutics.

脊髓背角的中枢敏化是神经病理性疼痛发生的重要机制。由于对中枢敏化的内在机制缺乏了解，因此，神经病理性疼痛的疼痛治疗十分困难。周围神经节TRPM3是一种新型伤害性热感受器。我们发现脊髓背角的TRPM3可能在神经病理性疼痛的中枢敏化以及治疗上起着非常重要的作用。基于这一发现，我们假设TRPM3可能通过调控PSD-95和NR2B的相互作用，介导了脊髓背角的中枢敏化，进而通过ERK和CREB磷酸化途径改变细胞的分化和重塑。本课题拟在大鼠神经病理性疼痛模型上，运用遗传学手段条件性过表达、采用在体RNA干扰沉默TRPM3基因，并结合动物行为学测定，研究TRPM3在病理性疼痛中的作用以验证以上假说；并且运用研究蛋白质相互作用的手段，探讨TRPM3与NR2B/PSD-95相互作用的关系，以揭示TRPM3介导中枢敏化和神经病理性疼痛的发病机制。该研究对未来发现治疗神经病理性疼痛有效手段具有重要意义。

慢性病理性疼痛始终困扰着人类，人群患病率约为10%–40%。脊髓背角是接受伤害性刺激和调制疼痛信号的初始驿站，对伤害性信号的处理直接影响着疼痛的发展和转归。研究发现，脊髓背角的中枢敏化是神经病理性疼痛发生的重要机制。但是，由于对中枢敏化的内在机制缺乏了解，神经病理性疼痛的疼痛治疗仍然十分困难。瞬时感受器电位M3（TRPM3）是一种热伤害性感受器，可能在中枢敏化中具有作用。而谷氨酸及其受体系统在脊髓背角中枢敏化中，扮演着不可替代的角色。研究发现，脊髓背角NR2B/PSD-95/CREB信号通路的激活与疼痛的发生有关。中枢神经系统NR2B与PSD-95具有相互作用，并且这种相互作用在慢性病理性疼痛的发生相关。基于此，我们假设TRPM3可能通过调控PSD-95/NR2B的相互作用，介导了脊髓背角的中枢敏化，进而通过ERK和CREB磷酸化途径改变细胞的分化和重塑。本课题拟在大鼠神经病理性疼痛模型上，运用药理学、遗传学和分子生物学手段TRPM3和谷氨酸受体信号在病理性疼痛中的作用，以揭示TRPM3介导中枢敏化和神经病理性疼痛的发病机制。经过4年的实施，已经基本完成了项目设计内容。有关研究结果发表于Pain Physician、Scientific Reports和Experimental Neurobiology等杂志。.我们首先采用免疫荧光和免疫印迹检测了TRPM3在大鼠脊髓背角的基础表达与分布规律。结果显示，大鼠脊髓背角TRPM3的基础表达较腹角丰富。免疫组化双标染色发现，TRPM3免疫反应阳性物与GFAP和Iba1没有共定位，而与NeuN具有部分共定位。我们发现干扰脊髓背角TRPM3，减轻了CCI大鼠对热刺激和机械刺激的痛觉过敏，为TRPM3参与病理性疼痛提供了有力的证据。我们发现，在大鼠CCI后，ACC的NR2B，PSD-95，Homer b/C的表达量显著性增加。提示谷氨酸信号通路的激活。ACC给予NR2B拮抗剂Ro 25-6981，减轻了大鼠由于CCI引起的对热刺激和机械刺激的痛觉过敏现象，并且抑制了PSD-95的表达，说明Ro 25-6981可能是通过下调PSD-95而产生镇痛作用。研究结果揭示了病理性疼痛脊髓背角中枢敏化的新机制，为慢性疼痛的预防和治疗提供了新的理论依据。